Trypanosoma brucei causes the disease African trypanosomiasis that afflicts an estimated 50,000-70,000 individuals with occasional pandemics resulting in deaths of up to 800,000 individuals. The limited number of drugs for treatment of African trypanosomiasis suffer from major deficiencies, including high toxicity, high cost, and development of drug resistance. Hence, there is a widely recognized urgent need for discovery of novel anti-trypanosomal drugs. Trypanosomes are critically dependent upon uptake and metabolism of glucose in the bloodstream form (BF) that causes disease, and the hexose transporter THT1 that is expressed in BF parasites has been identified and experimentally validated as an essential permease and highly promising drug target in multiple publications. Despite awareness of the promise of targeting THT1, essentially nothing has been done regarding identifying compounds that selectively inhibit this parasite permease. The novelty of this project is that it will carry out a screen for compounds that selectively inhibit THT1 and do not inhibit mammalian hexose transporters. These compounds can ultimately be employed to develop improved anti- trypanosomal drugs. A cell growth assay has been established and fully validated for high-throughput screening (HTS) to identify selective inhibitors of THT1. This assay employs a glucose transporter null mutant of the related parasite Leishmania mexicana that has been complemented with either the gene encoding THT1 or the gene for a major human glucose transporter GLUT1. In Aim 1, ~600,000 drug-like compounds from the St. Jude Children's Research Hospital CBT library will be screened for ability to inhibit growth of the transgenic L. mexicana expressing THT1. Hits from this primary screen will then be secondarily screened to select those that do not inhibit growth of transgenic L. mexicana expressing GLUT1. This two-step screen will identify compounds that are likely selective inhibitors of THT1. In Aim 2, these hit compounds will be tested in uptake assays employing [3H]D-glucose to identify those that are high affinity selective inhibitors of glucose uptake by THT1 but not by GLUT1. Compounds will also be tested for ability to inhibit growth of T. brucei BFs in vitro and in an established mouse model of African trypanosomiasis. Toxicity tests against several human cell lines will be performed to assess the therapeutic index of hit compounds, and metabolic stability will also be determined. This project takes advantage of an established collaboration between the laboratory of the PI Dr. Scott Landfear, a molecular parasitologist who has studied parasite transporters extensively, and that of Dr. Kip Guy, who operates an internationally renowned compound screening and pharmaceutical chemistry facility at St. Jude and who has a strong track record of screening for anti-parasitic compounds.